To achieve the long-term goal of restoring dental enamel, it is necessary to understand the fundamental chemical and biological principles of extracellular matrix assembly and the manner they control mineral nucleation and growth. There is still a large gap in our understanding of the underlying molecular mechanisms by which enamel matrix proteins assemble and interact with cells to control nucleation and oriented growth of hydroxyapatite crystals, and possibly cell movement and polarization. This is particularly true of ameloblastin protein, which is the focus of our proposed study. The goal of this proposal is therefore to advance our understanding of ameloblastin?s structure and function through a systematic investigation of its interactions with different targets. We hypothesize that the highly organized carbonated hydroxyapatite crystals in enamel continuously grow and form prismatic structures by means of complex ameloblastin-cell, ameloblastin- amelogenin, and ameloblastin-mineral interactions. Two major aims are proposed to systematically examine the above hypothesis by applying in vitro chemical models, cell culture and animal models for amelogenesis. Aim I: To investigate ameloblastin-cell membrane interactions and identify the interacting domains using in vitro (synthetic liposomes), and ameloblasts-like cell culture model systems. To design several mouse models with point mutations to the interacting domains identified, and examine the consequence of these mutations on enamel prismatic structure, ameloblasts morphology, and the attachment of the cells to the matrix. We hypothesize that ameloblastin interacts with ameloblasts cells via a domain in the sequence encoded by exon 5 and functions to anchor the mineralizing extracellular matrix to the enamel forming cells affecting cell polarization, migration and Tomes? processes formation. Aim II: To investigate amelogenin-ameloblastin interactions at a nanoscale level in vivo and in vitro and to identify their interacting domains using solution NMR spectroscopy. To study the dynamics of calcium phosphate mineralization events at a nanoscale level when ameloblastin is combined with amelogenin, using high resolution in situ atomic force (AFM) and transmission electron microscopy (TEM). We hypothesize that amelogenin and ameloblastin form hetereomolecular entities that are functional during different stages of amelogenesis. We anticipate to gain more insight into the structure, assembly properties and function of ameloblastin protein. We will define a novel cell- membrane- binding domain on ameloblastin protein. Specific protein-protein-interacting domains on the ameloblastin and amelogenin sequences will be identified. The effect of ameloblastin on mineralization will be elucidated and mineral-binding domains will be identified. These studies will advance understanding of the molecular function of ameloblastin in enamel biomineralization and will contribute to our efforts to fabricate synthetic enamel.